E. coli O157:H7 is an important pathogen derived from animal products or vegetables treated with animal waste. One of the factors that enables this organism to survive the stomach and colonize the large intestine of humans is its ability to tolerate acidic conditions. The mechanisms that confer acid tolerance are unclear, but probably colonization inhibition could be developed. Our overall goal for this proposal is to understand the role of acid 1) optimization of procedures for extracting E. coli O157:H7 DNA from bovine ruminal fluid and feces, 2) a 'shock' is necessary to induce the tolerance, 3) determination of the possible roles for rpoS (and other acid resistance systems) and anaerobiosis in allowing E. coli 0157:H7 to compete with microorganisms from the O157:H7 and ruminal lactate-utilizing bacteria as well as ruminal lactate-producing bacteria as well as ruminal lactate-producing bacteria. Our working hypothesis is that organisms that utilize lactate can assist in decreasing carriage of E. coli 0157:H7. It has been suggested that low ruminal pH of cattle consuming corn-based diets induces acid tolerance in E. coli O157:H7. However, this organism is capable of surviving in manure for long periods of time under environmental conditions. Thus, acid resistance may already be induced. The most promising plan of action appears to be reduction of E. coli O157:H7 carriage in cattle. Development of probiotic organisms that are indigenous to the rumen ecosystem holds the greatest potential for success. Thus, the long-term objective for our research is to develop probiotic treatment based on organisms indigenous to the rumen environment. The outcome of this study will assist others in future applications of this organism in management of infection by cattle, and hopefully in the protection of the food supply. By employing a host of techniques including anaerobic microbiology, PCR amplification, and hybridization, we are confident that we can realize the aims in this proposal within four years.